CN105648079B - A kind of osteoporosis blood testing target spot and its application - Google Patents
A kind of osteoporosis blood testing target spot and its application Download PDFInfo
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Abstract
The present invention relates to biomedicine fields, and in particular to a kind of osteoporosis blood testing target spot and its application more particularly relate to osteoporosis blood testing target spot CCDC91 and its application.Inventor screens the pathogenic target spot candidate gene CCDC91 of osteoporosis, and confirm that CCDC91 gene has good correlation with osteoporosis by molecular biology experiment, standby target spot is provided for clinical diagnosis, the efficient RNA interfering for CCDC91 is invented while being devised, is laid the foundation for the gene therapy in later period.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to a kind of osteoporosis blood testing target spot and its application, more
Body is related to osteoporosis blood testing target spot CCDC91 and its application.
Background technique
Osteoporosis (Osteoporosis) is characterized by ostosis reduction, bone resorption increase, and skeletal fragility increases
And the systemic disease of fragility fractures, main clinical manifestation are as follows: body pain, height cripetura, hunchback, especially the elderly easily occurs
Be easy fragility fractures, especially fractured near end of thighbone etc., bone density value decline be also at present osteoporosis diagnostic criteria it
One.Osteoporosis is with the increase at age, and disease incidence gradually increases, due to big, aging of population, ring by population base of China
The influence factors such as border pollution, Diet lifestyle variation, developing osteoporosis rate are in rise year by year trend, and age of onset has
Shifted to an earlier date.Osteoporotic fracture is senile osteoporosis most serious consequence, be easy to cause the elderly's marrow fracture, centrum pressure
The diseases such as contracting fracture, disease incidence is high, and operation risk is big, less effective, is that the elderly is caused to disable and lethal major reason
One of, and heavy financial burden is increased to society.
Two aspects of basic measure and drug therapy are broadly divided into the remedy measures of osteoporosis at present.Primary Care is arranged
Calcium agent and vitamin D are mainly supplemented in applying.Drug therapy mainly includes anti-bone resorption drug, Bone formation drug and its other medicine
Object.But since the pathogenesis to osteoporosis is not entirely clear that, so the treatment currently for osteoporosis is unsatisfactory.
Therefore, further research osteoporosis formed molecular mechanism and seek better treatment method, reduce incidence of fracture and
Improving the quality of living is the emphasis of current osteoporosis therapy research.
To solve the problems, such as that current osteoporosis molecular marked compound is rare, inventor is to 9 osteoporosis peripheral blood samples
And 6 Healthy People control peripheral blood samples carry out high-flux sequence, carry out the target spot base that causes a disease in conjunction with bioinformatics method analysis
The screening of cause picks out candidate gene CCDC91, further, experiments prove that CCDC91 gene and osteoporosis have very
Good correlation provides standby target spot for clinical diagnosis, invents while devising the efficient RNA interfering for CCDC91, after being
The gene therapy of phase lays the foundation.
Summary of the invention
The purpose of the present invention is to provide the albumen of CCDC91 gene and/or its expression to prepare anti-osteoporosis preparation
And/or the application in diagnosing osteoporosis preparation.
The purpose of the present invention is to provide detection CCDC91 genes and/or its preparation for expressing albumen in preparation osteoporosis
Application in diagnostic preparation.
Further, CCDC91 gene and/or its expression feelings for expressing albumen in diagnosing osteoporosis preparation detection peripheral blood
Condition.
Further, the diagnostic preparation of the osteoporosis includes being detected with fluorescence quantifying PCR method, method for gene chip
The expression of CCDC91 gene and/or CCDC91 albumen in peripheral blood.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker
Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Genetic chip is also known as DNA microarray (DNAmicroarray), can be divided into three kinds of main Types: 1) be fixed on poly-
The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
Hybridized by the target gene with fluorescent marker and is detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection
Technology, applied to the diagnosis of disease, advantage has the following aspects: first is that the sensitivity and accuracy of height；Second is that quickly
It is easy；Third is that a variety of diseases can be detected simultaneously.
The product for CCDC91 gene in fluorescence quantifying PCR method detection osteoporosis contains a pair of of specificity
Expand the primer of CCDC91 gene；The genetic chip includes the probe with the nucleic acid array hybridizing of CCDC91 gene.
Further, the diagnostic preparation of the osteoporosis includes the expression with immunization method detection CCDC91 albumen.It is excellent
Selecting CCDC91 protein expression in the immunologic detection method detection osteoporosis is western blot and/or ELISA and/glue
Body gold detection method.Preferably, the primer of a pair of of specific amplification CCDC91 gene, upstream primer sequence are SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.
Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detecting macromolecular antigen and specific antibody
Deng having many advantages, such as that quick, sensitive, easy, carrier is easy to standardize.ELISA detection kit is according to testing goal and operation
Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and
The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit
Enzyme (AP).
Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut
Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label,
So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized
Colloidal gold staining method for electron microscopy with the antibody of colloid gold label or antiantibody and negative staining Virus Sample or can be organized in conjunction with ultra-thin section,
Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made
Antigen or antibody point are first added sample to be examined on film by carrier after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) antigen of specificity or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold
Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added in the sample pad of test strips one end
Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to
When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
Further, the ELISA method of the detection CCDC91 albumen is to use ELISA detection kit.In the kit
Antibody commercially available CCDC91 monoclonal antibody can be used.Further, the kit includes: that coating CCDC91 monoclonal is anti-
The solid phase carrier of body, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate
Liquid etc..
Further, the colloidal gold method of the detection CCDC91 albumen is using detection kit, and the antibody can be used
Commercially available CCDC91 monoclonal antibody.Further, the gold-immunochromatographyreagent reagent for assay box using colloidal gold immunochromatographimethod technology or
Colloidal gold percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-
CCDC91 monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.
The purpose of the present invention is to provide a kind of PCR kit for fluorescence quantitative for detecting osteoporosis, which is characterized in that institute
Kit detection gene C CDC91 is stated, using special upstream primer and downstream primer, upstream primer sequence is SEQ ID
NO.1, downstream primer sequence are SEQ ID NO.2.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.Wherein the specific primer includes upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.The internal control primer is β-actin internal control primer, and upstream primer sequence is SEQ ID
NO.3, downstream primer sequence are SEQ ID NO.4.
The kit also includes RNA extraction agent.It is preferred thatReagent carries out sample rna extraction.
It is an object of the present invention to provide a kind of osteoporosis detection kit, the detection kit detects CCDC91 egg
It is white.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide it is a kind of detect osteoporosis genetic chip, the genetic chip include with
The probe of the nucleic acid array hybridizing of CCDC91 gene.
The purpose of the present invention is to provide CCDC91 genes and/or its protein inhibitor in preparing anti-osteoporosis preparation
Application.
To achieve the above object, the present invention passes through high-flux sequence combination bioinformatics method first and screens candidate base
Because of CCDC91, the relationship of CCDC91 and osteoporosis: CCDC91 and osteoporosis is then demonstrated by molecular biology method
It with good correlation, can be used for preparing anti-osteoporosis preparation and/or diagnosing osteoporosis preparation, there is important clinic
Application value.
Further, the anti-osteoporosis preparation refers to the preparation that can inhibit the expression of CCDC91 gene.People from this field
The expression of the known suppressor of member usually can be using one of following methods and/or several: passing through activation CCDC91 gene
Suppressor, activate CCDC91 gene inhibition of gene expression albumen, using RNA perturbation technique inhibit CCDC91 gene table
Reach, activate promote CCDC91 gene mRNA degradation microRNA, import promote CCDC91 gene coded protein degradation molecule,
Inhibit the factor of promotion CCDC91 gene expression and the expression of albumen.Pass through the suppressor of activation CCDC91 gene, activation
Inhibit the albumen of CCDC91 gene expression, import the siRNA for inhibiting CCDC91 gene expression, activation promotion CCDC91mRNA degradation
MicroRNA, import and promote the molecule of CCDC91 protein degradation, inhibit to promote the factor and albumen of CCDC91 gene expression
Expression.
RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo
Selective degradation, the phenomenon that leading to posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal
The expression of certain specific gene, promotes mRNA to degrade, and cells show is made to go out the technology of specific gene missing phenotype.SiRNA design
After the completion can be using direct synthesis technique or building SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate
The Mechanical Methods, cationic-liposome such as shallow lake method, electroporation, DEAE- glucan and polybrene method, microinjection or particle gun
The approach such as reagent method transfect cell.
Further, the siRNA target spot for inhibiting CCDC91 gene expression is selected from one of following sequence and/or several
Kind: SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11.It is preferred that siRNA sequence is SEQ ID NO.8.
Further, the siRNA sequence for inhibiting CCDC91 gene expression is selected from one of following sequence and/or several
Kind: SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.12, SEQ ID
NO.13.It is preferred that siRNA sequence is SEQ ID NO.9, SEQ ID NO.10.
The purpose of the present invention is to provide a kind of anti-osteoporosis preparation, the anti-osteoporosis preparation inhibits CCDC91 base
The expression of cause.
Further, using RNA interfering inhibit CCDC91 gene expression, RNA interfering target spot in following sequence one
It plants and/or several: SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, preferably SEQ ID NO.8.
Further, the siRNA sequence of CCDC91 gene expression is inhibited to be selected from one of following sequence and/or several: SEQ
ID NO.6,SEQ ID NO.7,SEQ ID NO.9,SEQ ID NO.10,SEQ ID NO.12,SEQ ID NO.13.It is preferred that
SEQ ID NO.9、SEQ ID NO.10。
Detailed description of the invention
Fig. 1 is CCDC91 gene relative expression's spirogram in osteoporosis peripheral blood and healthy human peripheral blood
Fig. 2 is each group CCDC91mRNA relative expression levels figure after RNA interference: C group: blank control group；C1 group: transfection rouge
Plastid group；C2 group: nonspecific siRNA group is transfected；S1, S2, S3 group: the siRNA group of specificity is transfected.
Specific embodiment
Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this
The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used
To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent
It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer
Part examinations.
1 high-flux sequence of embodiment and analysis
9 sufferers of osteoporosis face peripheral blood samples and 6 healthy human peripheral blood samples are collected respectively, carry out RNA extraction,
Agarose gel electrophoresis after RNA is extracted, whether the RNA sample that can be extracted from electrophoresis result with preliminary judgement is up-to-standard, if
It can be used for further transcriptome analysis.And then the extraction feelings of RNA sample are detected by NanoDrop1000 spectrophotometer
Condition, the sample requirement of RNA-seq sequencing: OD260/OD280 1.8-2.2.
Microarray dataset is the 2500 high-flux sequence platform of HiSeq of Illumina company, carries out high-throughput transcript profile depth
Sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/ after sequencing
Fastqc/) software carries out total evaluation, the quality Distribution value including base, the position point of mass value to the quality of sequencing data
Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.In differential genes expression analysis, according to obtaining
FPKM value, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, when screening, LOG2FC>1 or<-1, FDR<
0.05.In order to better understand the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene
Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above data
Analysis as a result, we have screened difference expression gene CCDC91 in conjunction with document.
CCDC91 expression conditions in 2 sufferers of osteoporosis face peripheral blood of embodiment and healthy human peripheral blood
One, material and method
1, material
74 sufferers of osteoporosis face peripheral bloods and 46 healthy human peripheral bloods are collected, it is grouped and is numbered.
2, method
The extraction of 2.1 osteoporosis peripheral bloods and healthy human peripheral blood total serum IgE
UsingReagent carries out sample rna extraction, and experimental implementation is carried out by product description.
RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.8-2.2；Total serum IgE electrophorogram has clearly
Clear 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
2.2 reverse transcription synthesizes cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcription, experimental implementation are carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid, following components are separately added into PCR pipe:
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
Aqua sterilisa is added to 25 μ l of total system in 2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid.42 DEG C are incubated for 1
Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservation.
2.3Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT method.
2.3.2 design of primers
Using online primer-design software, template sequence NM_018318 is closed by invitrogen company after design of primers
At.Specific primer sequence is as follows:
CCDC91 primer:
5’-TGAAGAATGGATGATGATG-3’(SEQ ID NO.1)
5’-GAATAGCAGGAGATGTTG-3’(SEQ ID NO.2)
Amplification length 94bp.
β-actin primer:
5’-TAATCTTCGCCTTAATACT-3’(SEQ ID NO.3)
5’-CCTTCATACATCTCAAGT-3’(SEQ ID NO.4)
Amplification length 103bp.
Operating process is as follows:
Reaction system: 2 × mix, 10 μ l；Upstream primer (10uM) and each 0.5 μ l of upstream and downstream primer (10uM)；2 μ l of template；
Sterile purified water filling-in is added to 25 μ l.
Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) is expanded, real
Operation is tested to carry out by product description.
Amplification program are as follows: 95 ° of 10min, (95 DEG C of 15sec, 55 DEG C of 60sec) × 45 circulations.
Sample RealTimePCR detection: 2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, uses target gene respectively
Primer and reference gene primer are expanded.Simultaneously in 60-95 DEG C of progress solubility curve analysis.
Two, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR, than
Compared with expression of the CCDC91 gene in sufferers of osteoporosis face peripheral blood and healthy human peripheral blood.(it is specifically shown in figure as the result is shown
1): qRT-PCR stable amplification result, wherein expression of the CCDC91 in osteoporosis peripheral blood is higher than healthy human peripheral blood
In expression, the former is about 3.5 times of the latter, and result above demonstrates the confluence analysis of high-throughput transcript profile expression data
CCDC91 highly expressed result in sufferers of osteoporosis face peripheral blood.
The culture of 3 cell line MG-63 of embodiment
One, experimental material
Cell line MG-63 is purchased from Chinese Academy of Sciences Shanghai cell research institute.
Two, main solution
Cell culture fluid :+10% standard fetal calf serum of DMEM culture medium.
0.25% tryptic digestive juice: 0.25g trypsase is added in 100m1 deionized water, filter filtration sterilization, point
Equipment is used.
Three, cell passes on
1 discards culture solution original in the culture bottle for covering with cell, and 0.25% trypsin solution 1m1 is added, and covering is thin
Born of the same parents' layer, bottleneck disinfection, covers；
Cellular change is observed under 2 inverted microscopes, over time, former adherent cell gradually tends to be round, carefully
Matrix retraction, space between cells increase, discard pancreatin in also non-levitating, and the culture solution that 5ml contains 10% fetal calf serum is added
Terminate digestion；
3 cell counts: taking above-mentioned cell suspension 0.5mI, instills in blood cell counting plate after appropriate dilution, based on leucocyte
The number big lattice inner cells in method number quadrangle four sum, when counting, only count nucleus and the complete cell of cytoplasm, and cell in heaps is pressed
One cell calculates, by the total number of cells in 4 block plaids by following formula scales at the cell number in every milliliter of cell suspension: thin
Big lattice total number of cells/4 × 10 born of the same parents' sum/ml=44× extension rate；
4 are further diluted to every milliliter containing 3 × 10 according to cell counts, with DMEM complete culture solution5A cell is dense
Degree, is sub-packed in culture bottle (every bottle of 8m1/), is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
Embodiment 4RNAi inhibits CCDC91 gene expression
One, experimental material
SiRNA building and synthesis
According to CCDC91 gene in GenBank (NCBI Reference Sequence:NM_018318) sequence design
Corresponding siRNA.Synesis Company's synthesis is sent to after design.Nonspecific siRNA is provided by Synesis Company.
Two, experimental method
(1) RNA perturbation technique specificity inhibits the expression of CCDC91 gene in MG-63 cell
1, the culture of MG-63 cell
Method and step is the same as embodiment 3.
2, the design and synthesis of siRNA
SiRNA expression vector pSIREN-DNR is marked containing neomycin resistance gene and GFP green fluorescence, can be monitored in real time
Transfection efficiency of the carrier in cell.According to purpose mRNA sequence, 3 RNA interference target sequence (table 1) are designed.Every is selected
Fixed siRNA target sequence designs siRNA positive-sense strand and antisense strand, is connected with loop (9nt), referred to as shRNA (short
hairpin RNA).Synthesize two single-stranded, single-stranded DNA doubles for obtaining shRNA of annealed dna of the DNA profiling of every coding shRNA
Chain template.Template strand followed by RNAPoIyIII polymerase transcription stop site, while both ends separately design BamHI and
HindIII restriction enzyme site can be cloned between BamHI the and HindIII restriction enzyme site of siRNA carrier multiple cloning sites.
After siRNA empty carrier BamHI and HindIII double digestion, 1% agarose gel electrophoresis recycles linear carrier.Annealing
DNA profiling double-strand is connected in linear carrier.Using T4 ligase, the molar ratio of insertion and carrier is about 3:1.Connection produces
Object converts DH5 α Escherichia coli, the coated plate on LB Amp culture medium, 37 DEG C of overnight incubations.PCR identification；Sequencing identification.Column extracting
Positive colony carrier is simultaneously quantitative.
1 siRNA transcription templates sequence of table
3, cell grouping and transfection
(1) cell is grouped
C group: blank control group；C1 group: transfection liposome group；C2 group: nonspecific siRNA group is transfected；S1, S2, S3
Group: the siRNA group of specificity is transfected.
(2) it transfects
According to LipofectamineTMThe step of 2000Transfection Reagent is provided carries out.
1. before transfection for 24 hours, the cell pancreatin of logarithmic growth phase is digested and is counted, dense with DMEM culture medium adjustment cell
Degree is 1 × 105/ ml takes 2m1 to be inoculated in six orifice plates, is placed in 37 DEG C, 5%CO2It cultivates in incubator, is merged in cell up to 80%
When for transfecting.With the DMEM culture medium culture 3-4h without serum before transfection.
2. preparing transfection liquid:
A liquid: 250u1 serum free medium dilutes 4.0ugDNA, mild to mix；
B liquid: 250u1 serum free medium dilutes 10u1Lipofectamine, mild to mix, and is placed at room temperature for 5min；
3. transfection: A liquid is mixed with B liquid, and compound is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate mixes gently.In CO2Liquid is changed in incubator after 37 DEG C of heat preservations 24-48h, 6h, the culture medium containing serum is added.
4, the verifying of transfection efficiency
(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope
After transfection for 24 hours, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence
Lower observation transfected condition.
(2) using the variation of Real-time PCR method detection transfection front and back CCDC91 gene expression
1. the building of standard curve: being chosen at 1 bottle of MG-63 cell normally cultivated in 50mI culture bottle, extract RNA, survey
Determine RNA concentration and purity, carry out reverse transcription reaction, ten times of the DNA profiling dilutions that reaction is generated obtain being equivalent to 104-
100The DNA profiling of copies/ul is separately added into CCDC91 primer and internal reference actin primer, prepares 25u1 reaction system, makes
With Real-time PCR amplification instrument, pcr amplification reaction is carried out.Obtain the standard curve of CCDC91 and actin.
2. the variation of Real-time PCR method detection transfection front and back CCDC91 gene expression: extracting group of cells
RNA measures RNA concentration and purity, carries out reverse transcription reaction, and every group of DNA profiling carries out the Real- of CCDC91 and actin simultaneously
Time PCR reaction, experiment is in triplicate.
3. carrying out agarose gel electrophoresis to PCR product.
Three, experimental result
Real-time PCR detects transfection efficiency.Using the mark of Real-time PCR method building CCDC91 and actin
Directrix curve, linear relationship is good, meets the requirements.Compare the expression of each group CCDC91 gene with the method for double standard curves.Blank
Control group, liposome transfection group, the expression of nonspecific transfection group gene are substantially similar, no significant difference.CCDC91-
SiRNA1, CCDC91-siRNA2, CCDC91-siRNA3 play the role of inhibiting CCDC91 gene expression, CCDC91-siRNA2
Effect become apparent from, inhibit efficiency up to 76%, and the inhibiting effect of CCDC91-siRNA1 and CCDC91-siRNA3 is respectively
42% and 29%, compared with blank control group, liposome transfection group, nonspecific transfection group, difference is statistically significant, and P <
0.05 (Fig. 2).
The present invention filters out osteoporosis pathogenic related gene CCDC91, binding molecule cell biological using high-flux sequence
Learn experimental verification, it was confirmed that CCDC91 has good correlation with osteoporosis.The present invention is osteoporosis clinic diagnosis
New target is provided, there is good potential applicability in clinical practice.
Claims (5)
1. the reagent of detection CCDC91 gene mRNA expression situation is preparing the application in diagnosing osteoporosis preparation.
2. application according to claim 1, which is characterized in that diagnosing osteoporosis preparation detects CCDC91 base in peripheral blood
Because of the expression of mRNA.
3. application according to claim 1, which is characterized in that the diagnosing osteoporosis preparation includes using fluorescent quantitation
The expression of PCR method, method for gene chip detection CCDC91 gene.
4. application according to claim 3, which is characterized in that with the production of fluorescence quantifying PCR method detection CCDC91 gene
Product contain the primer of a pair of of specific amplification CCDC91 gene；Genetic chip includes the nucleic acid array hybridizing with CCDC91 gene
Probe.
5. application according to claim 4, which is characterized in that the primer of the pair of specific amplification CCDC91 gene,
Upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101415836A (en) * | 2001-02-28 | 2009-04-22 | 基因信息公司 | Method for the detection of gene transcripts in blood and uses thereof |
| CN105132575A (en) * | 2015-09-28 | 2015-12-09 | 北京泱深生物信息技术有限公司 | Molecular marker for osteoporosis and application of marker |
| CN105256039A (en) * | 2015-10-29 | 2016-01-20 | 北京泱深生物信息技术有限公司 | Osteoporosis diagnosis marker |
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| CN101415836A (en) * | 2001-02-28 | 2009-04-22 | 基因信息公司 | Method for the detection of gene transcripts in blood and uses thereof |
| CN105132575A (en) * | 2015-09-28 | 2015-12-09 | 北京泱深生物信息技术有限公司 | Molecular marker for osteoporosis and application of marker |
| CN105256039A (en) * | 2015-10-29 | 2016-01-20 | 北京泱深生物信息技术有限公司 | Osteoporosis diagnosis marker |
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